A DNA sequence is described for the gene therapy of diseases associated with the immune system.
In its essential elements, the DNA sequence is composed of an activator sequence, a promoter module and a gene for the active substance.
The activator sequence is activated in a cell-specific or virus-specific manner and this activation is regulated by the promoter module in a cell cycle-specific manner. The choice of activator sequence and active substance depends on the indication area. The DNA sequence is inserted into a viral or non-viral vector, which vector is supplemented with a ligand having affinity for the target cell.
Depending on the choice of activator sequence and active substance, the following can be treated by administering the DNA sequence:
defective formation of blood cells
autoimmune diseases and allergies and, in addition, rejection reactions against transplanted organs
chronic arthritis
viral and parasitic infections and, in addition, prophylaxis of viral, bacterial and parasitic infections, and
leukemias.
A defective immune system causes an extremely wide variety of diseases. These include, for example,
allergies, autoimmune diseases and chronic inflammations, in particular chronic arthritis, due to erroneous functioning of the immune system
rejection of transplanted organs due to the immune system not being adequately inhibited
poor vaccination results and chronic infections, for example by viruses, as a consequence of immune deficiency
leukemias and lymphomas as tumorous degeneration of the immune system.
As is well known, the current therapeutic possibilities for diseases of this nature are inadequate. This will be illustrated using a few examples.
By now, a substantial number of cytokines and growth factors have become known which are involved in the differentiation, multiplication, maturation and functioning of cells.
For example, the hematopoietic system is controlled by a hierarchy of different cytokines, which ensure, by means of their differing functions, the multiplication of the individual differentiation stages and, over and above the individual differentiation stages, the ongoing formation of mature blood cells such as erythrocytes, thrombocytes, granulocytes, macrophages and lymphocytes (Dexter et al., Haematopoietic Growth Factors, Gardiner Well Communication, Macclesfield (1993)).
In addition, it is known that cytokines and growth factors play an important role in the cooperation of cells with each other (Pusztal et al., J. Pathol. 169, 191 (1993), Cross et al., Cell 64, 271 (1991)).
Thus, in immune resistance, for example, the collaboration between antigen-presenting cells, T lymphocytes and B lymphocytes is controlled by different cytokines with the sequence and concentration of the cytokines being crucial for the nature and strength of the immune reaction (Aulitzky et al., Drugs 48, 667 (1994), Sedlacek et al., Immune Reactions, Springer Verlag (1995)). In addition, resistance to infectious agents, such as viruses, is both influenced and supported by cytokines such as interferons (Edgington, Biotechnol. 11, 465 (1993)).
Knowledge of these relationships has already led to the development of cytokines for the therapy of human diseases, for example of
erythropoietin for curing anemia
G-CSF for curing neutropenia
GM-CSF for curing leukopenia
IL-2 for immune resistance to selected tumors
IFNxcex1 for the therapy of chronic viral hepatitis
IFNxcex2 for the therapy of multiple sclerosis
Further cytokines are currently being tested (Aulitzky et al., Drugs 48, 667 (1994)). These include, for example
thrombopoietin for curing thrombocytopenia (Metcalf, Nature 369, 519 (1994))
IL-3 for tumor therapy (de Vries et al., Stem Cells 11, 72 (1993) and for providing support in curing cytopenic conditions of the hematopoietic system (Freudl, Int. J. Immunopharm. 14, 421 (1992))
IL-4 for tumor therapy (Manate et al., Blood 83, 1731 (1994))
IL-6 for curing cytopenic conditions of the hematopoietic system (Brack et al., Int. J. Clin. Lab. Res. 22, 143 (1992))
IL-10 for immunosuppression (Benjamin et al., Leuk. Lymph. 12, 205 (1994))
IL-11 for curing thrombocytopenia (Kobayashi et al., Blood 4, 889 (1993)) IL-12 for tumor therapy (Tahara et al., Cancer Res. 54, 182 (1994))
TNFxcex1 for tumor therapy (Porter, Tibitech 9, 158 (1991)).
A common feature of therapy with all cytokines is the disadvantage that they usually have to be administered parenterally every day over a relatively long period of time and, furthermore, that, for their greatest possible efficacy, several cytokines either have to be injected one after the other in the necessary hierarchical sequence or corresponding cytokines have to be present in adequate concentration in the body.
That which is crucial for the effect is the concentration of the particular cytokines at the site of the cell which is to be stimulated. For the sake of simplicity, the cytokines are injected daily either subcutaneously or i.m. While this mode of administration guarantees a delayed systemic distribution, which is what is sought-after, relatively high quantities have to be administered in order to ensure an adequate local concentration at the site of the desired effect. The increased dose which is consequently required constitutes, due to the high level of expenditure involved in producing cytokines, a substantial cost factor which considerably restricts the use of cytokines.
Over and above this, some cytokines give rise, in the therapeutic dose range, to substantial side effects. IL-1 (Smith et al., New Engl. J. Med. 328, 756 (1993)), IL-3 (Kurzrock et al., J. Clin. Oncol. 9, 1241 (1991)) and I1-2 (Siegel et al., J. Clin. Oncol. 9, 694 (1991)) are examples of such cytokines.
Consequently, there is a substantial requirement for novel methods for making cytokines or combinations of cytokines available over a relatively long period of time, and in adequate concentration, at their site of action.
Despite improved antiinflammatory and immunosuppressive medicaments, chronic arthritis is a disease for which only inadequate therapeutic measures are available and which substantially reduces the quality of life and can even shorten life expectancy (Pincus et al., Bull. Rheum. Dis. 41, 1 (1992)). Because of its frequency (approx. 10% of the population of the western world suffers from arthritis) arthritis constitutes a substantial cost factor for national economies.
In view of the fact that medicinal therapy is inadequate, surgical removal of the synovial membranes of the joint capsule (synovectomy) or surgical replacement of the joint is the last possible form of therapy for many patients.
In view of these medicinal and economic problems, chronic arthritis represents a challenge for pharmaceutical research.
However, it can already be predicted today that, irrespective of their nature, medicaments which are administered orally or parenterally will have difficulty in reaching the region of joint inflammation in adequate concentration since they have to diffuse through the synovial capillaries and then passively through the synovial membrane into the joint cavity and from there into the cells lining the joint (Evans et al., Gene Therapeutics, J. Wolff, Editor, page 320, Birkhxc3xa4user, Boston 1994).
This diffusion is additionally made more difficult by the fact that the vascularization of the synovial membrane is significantly reduced in rheumatoid arthritis, for example (Stevens et al., Arthritis Rheum. 34, 1508 (1991)). While intraarticular injection of medicaments circumvents the problem of diffusion, the dwell time of the medicament in the joint is so short, owing to the high reabsorption rate, that repeated intraarticular injections over a relatively long period of time are required. These injections are in turn associated with the considerable risk of a joint infection. In addition, they can give rise to substantial side effects on account of the high local concentration of the medicament which is required.
In order to remedy these problems, the systemic or local, intraarticular administration of vectors or of in-vitro transduced synovial cells has been proposed for the therapy of chronic arthritis (Bandara et al., DNA Cell Biol. 11, 227 (1992), BBA 1134, 309 (1992) Evans et al., Transplant. Proc. 24, 2966 (1992)).
The principle of this gone therapy is that of using cells which are transduced in vivo in the joint cavity, or of using the injection into the joint cavity of synovial cells which have been transduced in vitro, to achieve high concentrations of antiarthritic substances, for example (Evans et al., J. Rheumatol. 21, 779 (1994))
antiinflammatory cytokines
(e.g. IL-1 receptor antagonist, IL-4 or IL-10)
cytokine inhibitors
(e.g. soluble receptors for IL-1, TNFxcex1, IL-8, TGFxcex1, or for other cytokines and interleukins which amplify inflammation)
enzyme inhibitors
(e.g. TIMP, LIMP, IMP, PAI-1, PAI-2 and others)
anti-adhesion molecules
(e.g. soluble CD-18, ICAM-1 and soluble CD44)
antagonists of oxygen radicals
(e.g. superoxide dismutase)
or of
growth factors for cartilage cells
(e.g. TGFxcex2 or IGF-1)
Animal experiments carried out in the rabbit have demonstrated grounds for IL-1-RA which is expressed following intraarticular injection of the corresponding gene having activity (Bandara et al., PNAS 90, 10764 (1993), Hung et al., Gene Therapy 1, 64 (1994)).
In principle, however, these methods for gene therapy which have hitherto been proposed suffer from considerable disadvantages
When synovial cells are transduced in vitro, they have to be removed from the joint cavity. This in itself puts a strain on the patient and carries the risk of a Joint infection. In the second place, synovial cells can only be isolated with great difficulty and in small numbers. Consequently, the synovial cells have to be replicated in vitro so that they can be transduced in sufficient number. However, it is known that it is only the fibroblast-like synovial cells (type B), and not the macrophage-like type A, which can be replicated under standard conditions of cell culture (Evans et al., Gene Therapeutics, page 320, J. A. Wolff, Editor, Birkxc3xa4user Boston (1994)). Consequently, the injection of synovial cells which are transduced in vitro suffers from substantial problems and will usually not be technically possible to achieve or only possible to achieve with considerable effort.
In the case of the systemic or intraarticular injection of vectors, which is under discussion, for transducing cells in vivo (Evans et al., Gene Therapeutics, page 320, J. A. Wolff, Editor, Birkxc3xa4user Boston (1994)), there is no regulatory mechanism which enables the genes which are transferred by way of the vector only to be expressed in those cells which are involved in chronic arthritis and then only if the cells are activated in the sense of an inflammation. In the absence of such a regulatory mechanism, cells which are distributed over the whole of the body are transduced, following systemic administration of the vector, to produce the particular antiarthritic substance, which would either lead to a systemic effect on the immune reaction or, in relation to the arthritic inflammatory process, be synonymous with the repeated systemic administration of antiarthritic active compounds, which administration is, per se, already regarded as being ineffective or insufficiently effective.
Following local administration, it would be possible, depending on the vector employed, to transduce in vivo either proliferating cells in the main (using an RTV vector) or resting cells as well (using other viral or non-viral vectors) to produce the antiarthritic substance. Since a large proportion of such substances have an antiinflammatory effect, the immune and inflammatory reactions in the joints would be inhibited independently of whether the chronic arthritis was in a resting phase or in an acute disease episode. Favored by the local inhibition of the immune reaction, and brought about by the causal factors of chronic arthritis, there would be the risk of an intensified pathological immune and inflammatory reaction once the activity of the antiarthritic substances had subsided, but no extensive alleviation or curing of the arthritis.
Consequently, there is a pressing requirement for novel therapeutic processes or active compounds
which can be administered locally or systemically to a patient depending on the number and severity of the chronically inflamed joints,
whose effect is principally, if not exclusively, restricted to activated and proliferating synovial cells or inflammatory cells,
whose effects primarily consist of the relatively long-term prophylaxis and therapy of the acute inflammatory episode.
Patients who have tumors of the hematopoietic system and who suffer a relapse after temporarily successful chemotherapy have a relatively poor prognosis (Hiddemann et al., Blood Rev. 8, 225 (1994)). As a consequence, various intensive treatment strategies have been developed for prolonging survival time.
These strategies include different combinations of cytostatic agents (The Medica Letter 31, 49 (1989)) and also bone marrow transplantation (De Magalhaos-Silverman et al., Cell Transplant. 2, 75 (1993)). However, the efficacy of both these approaches to therapy is only limited (Sloane et al., Histophathol. 22 201 (1993)). Consequently, there is still a substantial medical requirement for novel, effective therapeutic agents for tumors of the hematopoietic system.
Tumor cells of the hematopoietic system exhibit pronounced molecular biological changes which depend on the type of tumor (Reviews in Lotter et al., Cancer Surveys 16, 157 (1993) and Yunis et al., Crit. Rev. Onc. 4, 161 (1993)). The following are examples of those which are particularly pronounced in this context
Burkitt""s lymphomas (BL)xe2x80x94Deregulation of c-myc together with excessive production of c-myc mRNA and c-myc protein (McKeithan, Seminars in Oncol. 17, 30 (1990))
Overexpression of bcl-2 (Tsujimoto et al., PNAS 86, 1958 (1989))
B cell leukemias and lymphomas (BCL)
Overexpression of bcl-2 (in 85% of patients suffering from follicular lymphoma and 25% of patients suffering from diffuse lymphoma) (Yunis et al., New Engl. J. Med. 316, 79 (1987))
Overexpression of bcl-1 in patients suffering from centrocytic lymphoma (Seto et al., Oncogene 7, 1401 (1992))
Overexpression of IL-6 (Lewis et al., Nature 317, 544 (1985))
Overexpression of IL-10 (Levine, Blood 80, 8 (1992))
acute B cell leukemia (aBCL)
Expression of the fusion protein E2A-PBX-1 (Yunis et al., Crit. Rev. Onco. 4, 161 (1993))
T cell lymphomas (TCL)
Overexpression of c-myc (Cotter, Cancer Surveys 16, 157 (1993))
Overexpression of HOX11 (syn. TCL3) (Hatano et al., Science 253, 79 (1991))
chronic myeloid leukemia (CML)
Expression of the fusion protein BCR-Abl (Daley et al., PNAS 88, 11335 (1991))
acute lymphatic leukemia (ALL)
Overexpression of IL-3 (Mecker et al., Blood 76, 285 (1990))
acute myeloid leukemia (AML)
Expression of the fusion protein PML/RARA (Alcalay et al., PNAS 89, 4840 (1992)) Pandolfi et al., EMBO J. 11, 1397 (1992))
However, it has so far not been possible to use these molecular biological changes for clinical therapeutic methods.
The present invention now relates to an active compound (i.e. a pharmaceutical) which can be administered to patients both locally and systemically and which brings about a cell-specific, cell-cycle regulated formation of active substances for the therapy of diseases of the immune system.
An essential constituent of the active compound is a DNA construct of the following composition 
(In the whole of the text for this application, DNA is used as a common term both for a complementary (cDNA) and a genomic DNA sequence).
4.1. Description of the promoter module
The central element of the novel active compound is a cell cycle-regulated promoter module.
A cell cycle-regulated promoter module is to be understood, for example, to be the nucleotide sequence -CDE-CHR-Inr- (see below). The essential function of the promoter module moiety is that of inhibiting the function of the activator sequence in the G0/G1 phase of the cell cycle and that of ensuring cell cycle-specific expression in the S/G2 phase and consequently in proliferating cells.
The promoter module CDE-CER-Inr was discovered in the context of a detailed investigation of the G2-specific expression of the human cdc25C promoter. The starting point was finding a repressor element (cell cycle dependent element; CDR) which is responsible for switching off the promoter in the G1 phase of the cell cycle (Lucibello et al., EMBO U. 14, 132 (1995)). Using genomic dimethyl sulfate (DMS) footprinting and functional analyses (FIGS. 1 and 2), it was possible to demonstrate that the CDE binds a repressor (CDE-binding factor; CDF) in a G1-specific manner and in this way gives rise to conscription inhibition in non-proliferating (G0) cells. The CDE, which is located in the region of the basal promoter, depends, in its repressing function, on an upstream activating sequence (UAS). This led to the conclusion that the CDE-binding factor inhibits the transcription-activating effect of 5xe2x80x2-bound activator proteins in a cell cycle-dependent manner, i.e. in both non-proliferating cells and in the G1 phase of the cell cycle (FIG. 3).
It was possible to confirm this conclusion by a further experiment: fusion of the viral, non-cell cycle-regulated early Sv40 enhancer with a cdc25 minimum promoter (consisting of CDE and the start sites situated 3xe2x80x2) led to clear cell cycle-regulation of the chimeric promoter (FIG. 4). Subsequent investigations on the cdc25C enhancer have demonstrated that the transcription factors which are regulated by CDF in a cell cycle-dependent manner are NF-Y (CBF) (Dorn et al., Cell 50, 863 (1987), van Hijisduijnen et al., EMBO J. 9, 3119 (1990), Country et al., J. Biol. Chem. 270, 468 (1995)), Sp1 (Kadonaga et al., TIBS 11, 10 (1986) and a transcription factor (CIF) which is possibly novel and which binds to CBS7. Another interesting finding made in this study was the observation that NF-Y within the cdc25C enhancer only activates transcription efficiently in cooperation with at least one further NF-Y complex or with CIF. Both NF-Y and Sp1 belong to the glutamine-rich activator class, which provides important pointers to the mechanism of repression (e.g. interaction or interference with particular basal transcription factors or TAFs).
A comparison of the promoter sequences of cdc25C, cyclin A and cdc2 demonstrated homologies in several regions in several regions (FIG. 5, SEQ ID NOS 2-7). It is not only the CDE which is conserved in all three promoters (the divergencies which are present are not functionally relevant) but also the neighboring Yc boxes. As expected, all these regions exhibited protein binding in vivo, with this protein binding being cell cycle-dependent in the came of the CDE. In addition, it was demonstrated that all 3 promoters are deregulated by mutation of the CDE (Table 1). When the cdc25C, cyclin A and cdc2 sequences were compared, it was clear that there was also a remarkable similarity in the region immediately 3xe2x80x2 of the CDE (cell cycle genes homology region; CHR) (FIG. 5, SEQ ID NOS 2-7). Although this region is functionally as important as CDZ (Table 1), it is not visible in the in-vivo DMS footprinting experiments. A possible explanation to this is an interaction of the factor with the minor groove of the DNA. Results from electrophoretic mobility shift assay (ENSA) experiments indicate that CDE and CHR jointly bind a protein complex, the CDF. These observations indicate that the CDF-mediated repression of glutaminerich activators is a frequently occurring mechanism in cell cycle-regulated transcription.
However, it is apparently not only the CDE-CHR region which is of importance for regulating the cdc25C promoter but also one of the initiation sites (position +1) within the nucleotide sequence of the basal promoter (positions xe2x89xa6xe2x88x9220 to xe2x89xa7+30, see FIG. 1, SEQ ID NO:1). Mutations in this region, which includes the in-vitro binding site for YY-1 (Seto and Shenk, Nature 354, 241 (1991), Usheva and Shenk, Cell 76, 1115 (1994)) lead to complete deregulation. In view of the proximity of the CDE-CHR to the basal promoter, it is consequently very probable that the CDF interacts with the basal transcription complex.
4.2. Description of the activator sequence
An activator sequence (UAS=upstream activator sequence) is to be understood to be a nucleotide sequence (promoter sequence or enhancer sequence) with which transcription factors, which are formed or are active in the target cell, interact. The CMV enhancer, the CMV promoter (EP 0173. 177.B1), the SV40 promoter, or any other promoter sequence or enhancer sequence known to the skilled person, can be used as an activator sequence.
Within the meaning of this invention, however, the preferred activator sequences include gene-regulatory sequences or elements from genes which encode proteins which are formed, in particular, in cells of the hematopoietic system, in activated lymphocytes, in activated synovial cells or macrophages, in virus-infected cells or in leukemia cells.
4.3. Description of the active substance
The active substance is to be understood to be the DNA for a protein which is to bring about the therapeutic effect, i.e. the curing of the disease of the immune system, and to the site of formation. The choice of the nucleotide sequences for the activator sequence and the active substance depends on the target cell and the active substance which is desired.
4.4. Preparation of the plasmid or vector
The novel DNA construct is made into a complete vector in a manner with which the skilled person is familiar; thus, for example, it is inserted into a viral vector (in this regard, see D. Jolly, Cancer Gene Therapy 1, 51 (1994)), or used as a plasmid. Viral vectors or plasmids can be complexed with colloidal dispersions, for example with liposomes (Farhood et al., Annals of the New York Academy of Sciences 716, 23 (1994)) or with a polylysine/ligand conjugate (Curiel et al., Annals of the New York Academy of Sciences 716, 36 (1994)).
4.5. Supplementation with a ligand
Viral or non-viral vectors of this nature can be supplemented with a ligand which has binding affinity for a membrane structure on the selected target cell. The choice of the ligand consequently depends on the choice of the target cell.